Oncogene (2012), 1–9 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc

ORIGINAL ARTICLE The deubiquitinating enzyme USP37 regulates the oncogenic fusion PLZF/RARA stability

W-C Yang1,2,3 and H-M Shih1,2,3

Acute promyelocytic leukemia (APL) is predominantly characterized by chromosomal translocations between the retinoic acid receptor, alpha (RARA) and the promyelocytic leukemia (PML) or promyelocytic leukemia finger (PLZF) gene. In APL cells with PML/ RARA fusions, arsenic trioxide and all-trans retinoic acid treatments specifically target the fusion protein for -dependent degradation, thereby promoting cellular differentiation and clinical remission of disease. In contrast, APL cells expressing PLZF/RARA fusion are largely resistant to similar treatments and prognosis for patients with this translocation is poor. Understanding the molecular mechanisms regulating PLZF/RARA protein stability would provide novel therapeutic targets for PLZF/RARA-associated APL. Toward this end, we have performed an RNAi-based screen to identify factors affecting PLZF/RARA stability. Among the factors identified was the -specific peptidase 37 (USP37). We showed that USP37 interacted with PLZF/RARA through the PLZF moiety and sustained PLZF/RARA steady state levels. Domain mapping study revealed that N-terminal domain of USP37 is required for the PLZF/RARA interaction and protein regulation. Furthermore, overexpression or depletion of USP37 caused an increase or decrease of PLZF/RARA protein half-life, correlating with down- or upregulation of PLZF/RARA poly-ubiquitination, respectively. By PLZF/RARA- transduced primary mouse hematopoietic progenitor cells, we demonstrated that Usp37 knockdown alleviated PLZF/RARA-mediated target gene suppression and cell transformation potential. Altogether, our findings of USP37-modulating PLZF/RARA stability and cell transformation suggest that USP37 is a potential therapeutic target for PLZF/RARA-associated APL.

Oncogene advance online publication, 3 December 2012; doi:10.1038/onc.2012.537 Keywords: acute promyelocytic leukemia; deubiquitinating enzyme; USP37; PLZF; RARA

INTRODUCTION Ubiquitination-proteasome-dependent is a major 9 Acute promyelocytic leukemia (APL) is a rare disease characterized cellular pathway to control protein stability. Protein ubiqui- by the chromosomal translocations between the retinoic acid tination is a cascade reaction involving a group of specialized receptor, alpha (RARA) gene and its counterpart gene (X), resulting protein family called ubiquitin-activating enzyme E1, ubiquitin- in an aberrant fusion protein X-RARA, such as promyelocytic conjugating enzyme E2 and E3. Conversely, this leukemia (PML)/RARA, promyelocytic leukemia zinc finger (PLZF)/ biological process can be reversed by deubiquitinating enzymes RARA, NPM/RARA, NuMA/RARA or STAT5b/RARA.1 In APL patients, (DUBs), which are functioning by removing conjugated abnormal accumulation of undifferentiated promyelocytes is ubiquitin from substrates.10 In general, the ubiquitination level of generally observed in bone marrow, which is because of the a substrate is regulated by its associated E3 ubiquitin ligase blockage of cellular differentiation in myeloid lineage. Such and/or DUB, correlating with the regulation of substrate protein differentiation arrest is in part resulted from dysregulation of stability. Thus, E3 ubiquitin ligases and DUBs are considered as key transcriptional regulators such as CEBPa involved in myeloid potential targets for regulation of disease-associated protein differentiation by those X-RARA proteins.2,3 All-trans retinoic acid stability. By sequence comparison, about 100 DUB have (ATRA) treatment has been introduced for APL cells expressing been annotated in genome.11,12 According to the protein those X-RARA fusions by promoting cellular differentiation and secondary structure of DUBs, these DUBs can be divided into five clinical remission of disease. Although APL cells expressing PLZF/ different subclasses, including four DUBs, RARA fusion are responsive to ATRA treatment for cell ubiquitin-specific protease (USP), Machado–Joseph disease pro- differentiation,4 PLZF/RARA-associated APL patients, distinct tease, Otubain protease (OTU), and -terminal hydrolase, from other X-RARA types of APL patients, are resistant to ATRA and one metalloprotease DUB, JAB1/MPN/Mov34 metalloen- therapy.2 In addition to ATRA, arsenic trioxide has been zyme.12 By global proteomic approach, DUBs have been shown successfully used for clinical remission of APL patients carrying to participate in several cellular functions, including DNA damage PML/RARA fusions specifically.5,6 Arsenic trioxide treatment and repair, protein quality control and degradation, RNA specifically triggers the poly-SUMO chain formation of PML/ transcription and processing, and signal transductions.13 RARA, subsequently targeting PML/RARA fusions for poly- In present study, we have identified USP37-regulating PLZF/ ubiquitination and protein degradation.7,8 Such therapy- RARA protein stability by RNAi screening. USP37 could bind and triggered degradation of an oncoprotein could potentially serve deubiquitinate PLZF/RARA fusion, thereby enhancing PLZF/RARA as a general strategy to eliminate cancer cells. protein stability. Knockdown of Usp37 in mouse primary

1Molecular Medicine Program, Taiwan International Graduate Program (TIGP), Academia Sinica, Taipei, Taiwan; 2Institute of Biochemistry and Molecular Biology, School of Life Science, National Yang-Ming University, Taipei, Taiwan and 3Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. Correspondence: Professor H-M Shih, Institute of Biomedical Sciences, Academia Sinica, 128, Sec.2, Academia Rd., Nankang 115, Taipei, Taiwan. E-mail: [email protected] Received 30 May 2012; revised 3 October 2012; accepted 19 October 2012 USP37 deubiquitinates PLZF/RARA W-C Yang and H-M Shih 2 hematopoietic progenitor cells could attenuate PLZF/RARA- RARA/PLZF (Figure 2b), neither affected the protein level of mediated target gene suppression and cell transformation. These another abnormal fusion protein PML/RARA (Figure 2c). In results suggest that USP37 is an important factor in modulating contrast, expression of USP29 but not OTUD7B could increase oncogenesis induced by PLZF/RARA fusion in APL cells. PML/RARA protein level (Supplementary Figure S2c). These data suggest that USP37 may stabilize PLZF/RARA through the PLZF moiety, whereas USP29 may exert similar function via the RARA RESULTS moiety in cells. The nature of OTUD7B affecting the levels of PLZF/ RNAi screening identifies DUBs modulating PLZF/RARA protein RARA and RARA, but not the levels of PLZF and PML/RARA, is level currently unclear. We further substantiated the specificity of To monitor PLZF/RARA protein level in cells, we have generated USP37 targeting to the PLZF moiety by USP37 knockdown the U937 myeloid leukemia cell line carrying a cassette in which experiments. USP37 depletion reduced endogenous PLZF level both EGFP-fused PLZF/RARA and tRFP driven by an internal but not RARA level in HL60 cells (Figure 2d, left panel). In contrast, entry site were induced to express by addition of USP37 knockdown failed to significantly alter the protein level of doxycycline (Figure 1a). In this screening, EGFP intensity was endogenous PML/RARA and RARA in NB4 cells (right panel), a cell measured to represent PLZF/RARA protein expression level, while line derived from long-term cultures of human APL. Altogether, tRFP intensity was served as an internal control for the normal- the findings that USP37 conferred the specific regulation on PLZF/ ization of EGFP intensity. Lentiviruses carrying shRNA clones RARA, but not on PML/RARA level, led us to focus on the study of targeting to 83 human DUBs were used to infect U937 cells USP37 in regulating PLZF/RARA. (Supplementary Table S1). Following the puromycin selection for We next examined whether USP37-regulated PLZF/RARA cells expressing shRNA and doxycycline induction for expression protein level is relevant to its protease catalytic activity. USP37 of the PLZF/RARA cassette, the cells were subjected to 96-well catalytically inactive mutant, converting Cys350 to Ala (CA), was flow cytometry analysis for measuring both EGFP and tRFP generated to examine for PLZF/RARA regulation. As expected, the intensities (Figure 1b). The P-values were calculated and adjusted USP37 CA mutant was impaired to enhance PLZF/RARA protein on the basis of comparing EGFP/tRFP ratio between knockdown level as compared with WT (Figure 2e), indicating the importance and control clones. To avoid the possible off-target effects of of USP37 catalytic activity for PLZF/RARA regulation. Noted certain shRNA clones, DUB targeted by at least three shRNA clones that overexpression of USP37 CA mutant rendered USP37 showing significant effect (P-valueo0.0001) on decreasing multiple-band shifts. These slowly migrating bands were K48- EGFP/tRFP ratio was chosen as a candidate for further study. linkage ubiquitinated USP37 proteins, as evidenced by western Eight candidate DUBs, including MPND, OTUD5, OTUD6A, blot analysis with antibodies against specific ubiquitin linkage OTUD6B, OTUD7B, USP2, USP29 and USP37 were initially identified (Supplementary Figure S3). In addition, PLZF/RARA mRNA (Supplementary Table S2). To further validate whether level was not altered by USP37 overexpression (Supplementary these candidate DUBs regulate PLZF/RARA protein expression, Figure S4), suggesting that the regulation of PLZF/RARA by USP37 each candidate DUB was ectopically expressed with PLZF/RARA in was not at the mRNA level. HEK-293T cells. Only OTUD6A, OTUD7B, USP2, USP29 and USP37 could enhance PLZF/RARA protein expression in a dosage-dependent manner (Figure 1c), suggesting that these are USP37 regulates PLZF/RARA protein level via a direct protein interaction potential DUBs to regulate PLZF/RARA protein level. The discrepancy between knockdown and overexpression of these Given that USP37 regulates PLZF/RARA protein steady-state level, MPND, OTUD5 and OTUD6B in affecting PLZF/RARA protein we further assessed whether USP37-regulated PLZF/RARA protein steady-state level is currently unclear. It is possible that distinct level is through a protein interaction between USP37 and PLZF/ cellular contexts of HEK-293T and U937 cells may contribute to RARA. To this end, we first demonstrated that USP37 and PLZF/ such a discrepancy. RARA can form complexes in cells. The results of co-immunopre- cipitation experiments revealed that overexpressed Flag-tagged USP37 could be detected in the immunocomplex of HA-PLZF/ USP37 modulates PLZF/RARA protein level through PLZF moiety RARA (Figure 3a), and the interaction of USP37 and PLZF/RARA Because PLZF/RARA proteins are mainly localized in the nuclear was further confirmed by reciprocal immunoprecipitation experi- compartment, we next examined the subcellular localization of ments (Figure 3a). We further substantiated the complex these five candidate DUBs for possible direct interaction and formation of PLZF/RARA with endogenous USP37, using U937 regulation of PLZF/RARA. The results of immunofluorescence and cells conditionally expressing Flag-tagged PLZF/RARA by adding western analyses demonstrated that those candidate DUBs, doxycycline. As expected, endogenous USP37 could form com- including USP29, USP37, OTUD6A and OTUD7B, were present in plexes with PLZF/RARA in cells by the immunoprecipitation with the nuclear compartment (Supplementary Figures S1a and b). Of either anti-USP37 or anti-Flag antibody (Figure 3b). To further test note, OTUD6A was distributed in the peri-nuclear region whether USP37 binding to PLZF/RARA is through a direct (Supplementary Figure S1a). These results implicated possible interaction, in vitro GST pull-down assay was performed using involvement of USP29, USP37 and OTUD7B in PLZF/RARA GST-fused PLZF/RARA and MBP-fused USP37 recombinant pro- regulation via a direct association. teins. Notably, MBP-USP37 could be pulled down by GST-PLZF/ Because PLZF/RARA is a fusion protein resulted from reciprocal RARA but not by GST (Figure 3c), suggesting that USP37 can chromosomal translocations between PLZF and RARA genes, it is directly interact with PLZF/RARA. possible that USP29, USP37 or OTUD7B could modulate PLZF/ To demonstrate the importance of USP37-PLZF/RARA interac- RARA protein level through the moiety of PLZF or RARA, or both tion for PLZF/RARA protein regulation, we performed the domain portions. To test these possibilities, HA-tagged PLZF or RARA was mapping study. In line with the result that USP37 enhanced PLZF transiently co-expressed with Flag-tagged USP29, USP37 or but not RARA protein steady-state level (Figure 2a), Flag-tagged OTUD7B in HEK-293T cells. Of note, PLZF protein but not RARA USP37 could precipitate HA-tagged PLZF but not HA-tagged RARA protein was elevated by USP37 in a dose-dependent manner (Figure 3d). On the front of USP37, we generated and tested N- (Figure 2a), while expression of USP29 or OTUD7B enhanced RARA and C-terminal deletion mutants of USP37 for PLZF/RARA protein but not PLZF protein (Supplementary Figures S2a and b). interaction (Figure 3e). The results of co-immunoprecipitation Furthermore, increasing USP37 expression was unable to alter the experiments showed that N-terminal deletion mutant (301–979), protein level of the reciprocal chromosomal translocation product, although it contains the entire USP domain, significantly reduced

Oncogene (2012), 1 – 9 & 2012 Macmillan Publishers Limited USP37 deubiquitinates PLZF/RARA W-C Yang and H-M Shih 3

Figure 1. Identification of candidate DUBs in regulating PLZF/RARA protein level. (a) A schematic diagram of the cassette construct expressing EGFP-PLZF/RARA and tRFP. (b) Flowchart of functional RNAi screening for regulating PLZF/RARA level. (c) Western blots show the PLZF/RARA levels in HEK-293T cells cotransfected with indicated DUB constructs along with CMV-EGFP. EGFP was used as cotransfection control. the PLZF/RARA interaction (Figure 3e, lane 4), while USP37 steady-state level of USP37 (301–979) fragment was not C-terminal deletion mutant (1–700) bound to PLZF/RARA with significantly affected by USP37, compared with (1–700) fragment the extent slightly lesser to WT (lanes 2 and 3). Of note, the protein or WT (Figure 3f). Altogether, these results provide a nice

& 2012 Macmillan Publishers Limited Oncogene (2012), 1 – 9 USP37 deubiquitinates PLZF/RARA W-C Yang and H-M Shih 4

Figure 2. USP37 regulates PLZF/RARA expression level. (a-c) Western blots of HEK-293T cells transfected with Flag-tagged USP37 and EGFP along with HA-tagged PLZF or RARA (a), or HA-tagged RARA/PLZF (b) or HA-tagged PML/RARA (c). (d) Immnoblotting shows endogenous level of USP37, PLZF, RARA and PML/RARA in indicated cells with shUSP37 or shLuc. Arrow indicates PML/RARA. (e) Immunoblots show PLZF/ RARA levels in HEK-293T cells cotransfected with USP37 WT or catalytic mutant C350A (CA). Arrowhead indicates non-modified band of USP37 CA mutant.

correlation between the binding and regulation of PLZF/RARA USP37 modulates the cell transformation potential of PLZF/RARA protein by USP37. PLZF/RARA is able to transform hematopoietic progenitor cells by increasing capacity of cell self-renew and proliferation, and blocking the differentiation of myeloid cell lineage.14 Because USP37 regulates the protein stability and ubiquitination of PLZF/ USP37 regulates PLZF/RARA protein stability, it is conceivable that RARA USP37 affects PLZF/RARA-mediated cell transformation of primary We next examined the effect of USP37 on PLZF/RARA protein hematopoietic progenitor cells. To test this possible scenario, we half-life. cycloheximide (CHX)-chase experiments showed that first established the PLZF/RARA-mediated transformation using USP37 WT, but not CA mutant, prolonged PLZF/RARA protein mouse primary hematopoietic progenitor cells transduced by half-life in HEK-293T cells (Supplementary Figures S5a and b). retrovirus-expressing PLZF/RARA. As a control, PLZF/RARA- Likewise, knockdown of USP37 by shRNA clones identified from transduced hematopoietic progenitor cells showed decreased initial RNAi screening (Supplementary Figure S5c and Supple- expression levels of CCAAT/enhancer-binding protein family mentary Table S2) showed a reduction of PLZF/RARA protein half- members including Cebpa, Cebpb and Cebpe, compared with life in U937 cells (Supplementary Figures S5d and e). These results cells infected with retrovirus carrying an empty vector (Figure 5a). clearly demonstrate that USP37 enhances PLZF/RARA protein These results are consistent with previous reports showing stability. suppression of CCAAT/enhancer-binding protein transcriptional In addition, the findings that USP37 catalytic activity is required factors involved in APL cells.3,15 for increasing PLZF/RARA protein stability led us to test whether We next accessed the effect of Usp37 on PLZF/RARA-mediated USP37 modulates PLZF/RARA protein ubiquitination level. Over- transformation by knockdown experiments. We infected the PLZF/ expression of USP37 WT, but not CA mutant, significantly RARA-transduced cells with lentivirus-expressing Usp37 shRNAs. decreased the ubiquitination of PLZF/RARA in HEK-293T cells Two different Usp37 shRNAs were tested for depletion efficiency. (Figure 4a). It should be noted that effects of USP37 on PLZF/RARA The shUsp37#2 showed a better efficiency in downregulating deubiquitination were specific because global ubiquitination Usp37 than the shUsp37#1 (Figure 5b, left panel). As expected, profile was not significantly changed by USP37 overexpression depletion of Usp37 expression alleviated PLZF/RARA-associated (Supplementary Figure S6). Accordingly, knockdown of USP37 in suppression of CCAAT/enhancer-binding protein family gene U937 cells increased the ubiquitination level of PLZF/RARA expression (Figure 5b, right panel). Consistent with the depletion (Figure 4b). We further demonstrated that the recombinant efficiency, we observed that shUsp37#2-treated cells yielded USP37 WT but not CA mutant could deubiquitinate PLZF/RARA higher levels of the CCAAT/enhancer-binding protein family in vitro (Figure 4c). Along with above binding study results, these gene expression than shUsp37#1-treated cells. These results data strongly suggest that USP37 enhances PLZF/RARA protein suggest that Usp37 is important for PLZF/RARA-mediated cell stability by deubiquitinating PLZF/RARA. transformation.

Oncogene (2012), 1 – 9 & 2012 Macmillan Publishers Limited USP37 deubiquitinates PLZF/RARA W-C Yang and H-M Shih 5

Figure 3. USP37 interacts with PLZF/RARA. (a) Western blots show the complex formation of Flag-tagged USP37 and HA-tagged PLZF/RARA in HEK-293T cells transfected with indicated constructs. (b) Western blots show the interaction of endogenous USP37 and Flag-tagged PLZF/ RARA in TetOn-U937 cells with or without 0.5 mg/ml doxycycline induction for 16 h and precipitated by anti-USP37 or anti-Flag antibody. (c) Immunoblotting shows USP37 pulled down by GST-PLZF/RARA. Input represents the 10% amount of recombinant USP37 protein subjected to binding assays. Coomassie blue staining shows GST fusion proteins used for each binding reaction. (d) Western blots show the complex formation of USP37 with PLZF but not with RARA in HEK-293T cells transfected with indicated constructs. Asterisk indicates IgG heavy chain from immunoprecipitation. (e) Diagram view of wild-type and deleted mutants of USP37. The USP domain is indicated. Western blotting analysis of immunoprecipitated complex from HEK-293T cells transfected with indicated constructs. (f) Western blots show the PLZF/ RARA levels in HEK-293T cells cotransfected with USP37 WT or deletion mutants.

To demonstrate the role of Usp37 in PLZF/RARA-elicited and 4). Of note, Usp37 depletion failed to reduce PML/RARA- transformation, we further performed the methylcellulose culture elicited hematopoietic cells grown in methylcellulose (Supple- of mouse primary hematopoietic progenitor cells transduced by mentary Figure S7). These data suggest that the effect of Usp37 PLZF/RARA along with or without Usp37 knockdown.16 As a knockdown on the reduction of PLZF/RARA-induced clonogenicity control, hematopoietic progenitor cells infected by retrovirus is the consequence of PLZF/RARA loss. carrying MSCV empty vector showed a loss of colony-formation In line with the colony-formation number, we observed that ability after the second passage in methylcellulose culture PLZF/RARA expression induced large and compact colony (Figure 5c, lanes 1 and 2). Notably, these cells expressing either formation on methylcellulose culture (Figure 5d, panels i and ii). shLuc or shUsp37#2 gave comparable colony number (Figure 5c, Usp37 knockdown changed PLZF/RARA-elicited colony-formation lanes 1–4), indicating Usp37 depletion did not alter the pattern from compact and large to diffuse and smaller in size characteristics of hematopoietic progenitors in methylcellulose (panel ii versus iv). Furthermore, Giemsa staining showed that culture. By contrast, PLZF/RARA-transduced cells conferred a PLZF/RARA-transduced hematopoietic progenitor cells yielded significant increase of colony number after the second replating immature cells with a feature of large nucleus and scant cytoplasm (lane 6). Remarkably, such PLZF/RARA-mediated colony formation as compared with control cells (Figure 5e, panels i and ii). Of note, was significantly reduced in Usp37-depeleted cells (lane 8) to an such PLZF/RARA-elicited immature cell phenotype was signifi- extent close to that of cells infected with control vector (lanes 2 cantly decreased in Usp37 knockdown cells (panel iv). Accordingly,

& 2012 Macmillan Publishers Limited Oncogene (2012), 1 – 9 USP37 deubiquitinates PLZF/RARA W-C Yang and H-M Shih 6

Figure 4. USP37 deubiquitinates PLZF/RARA. (a) Western blots show PLZF/RARA deubiquitinated by USP37 in HEK-293T cells transfected with indicated constructs and treated with 20 mM MG132 for 4 h before harvest. (b) Immunoblotting shows PLZF/RARA ubiquitination in TetOn- U937 cells with USP37 knockdown. TetOn-U937 cells expressing FLAG-PLZF/RARA were infected with lentivirus carrying shLuc or shUSP37#1 construct and treated with 10 mM MG132 for 4 h before harvest. (c) Immunoblotting shows in vitro deubiquitination of poly-ubiquitinated PLZF/RARA immunoprecipitated from HEK-293T cell lysates incubated with purified USP37 WT or CA mutant protein for 2 h. Input represents the 10% amount of immunoprecipitated proteins subjected to deubiquitination assays. Coomassie blue staining shows USP37 proteins used for each deubiquitinating reaction.

FACS analyses revealed that PLZF/RARA-transduced hematopoie- phosphorylates USP37 and enhances its deubiquitinating activity tic progenitor cells rendered a marked increase of the c-kit toward cyclin A, forming a positive feedback loop to promote S progenitor cell marker together with a reduction of myeloid phase entry. Interestingly, PLZF/RARA was also shown to activate differentiation markers Gr-1 and Mac-1 (Figure 5f, panels ii and vi), the expression of cyclin A1,23 an alternative CDK2 associated A-type compared with control cells (panels i and v). Such poor cyclin, in human hematopoietic progenitor cells.24 Since cyclin A1 differentiation phenotypes were significantly attenuated by could form complex with CDK2 for substrate phosphorylation and Usp37 knockdown (panels iv and viii). Noted that Usp37 also contribute to G1-S cell cycle progression in somatic cells,25 it is knockdown did not significantly change the myeloid cell possible that PLZF/RARA-mediated transformation of hemato- differentiation profiles (panels iii and vii), implying that Usp37 poietic progenitor cells is in part via activation of cyclin A1-CDK2/ itself is not associated with normal hematopoietic progenitor USP37 positive loop. In addition to causing PLZF/RARA protein cell differentiation into myeloid lineage. Altogether, our data destabilization, USP37 knockdown may also destroy this positive suggest that USP37 has an important role in regulating the protein feedback loop, thus significantly reducing PLZF/RARA-mediated stability and transformation capacity of PLZF/RARA in myeloid cell transformation (Figure 5). lineage. Besides the involvement of USP37 in PLZF/RARA-mediated cell transformation, our results that USP37 targeting to PLZF/RARA is through its N-terminal domain and PLZF moiety also implicate DISCUSSION that USP37 may play a role in modulating PLZF protein level in a While the substrates and biological functions of most DUBs are physiological context. Recent studies reported that PLZF partici- unclear, accumulating evidences indicate that DUBs are potential pates in several biological functions, including a negative role in important targets for the treatment of human diseases, such as myeloid cell differentiation26–28 and a role in male germ cells for cancer. For example, USP7 has been shown to stabilize , stem cell-renewal.29,30 We found that USP37 knockdown could causing tumor suppressor p53 degradation.17 USP9X was reported enhance HL60 granulocytic differentiation induced by ATRA, to deubiquitinate and stabilize MCL1 in varied human cancer cells, similar to the extent of HL60 cells with PLZF depletion thus maintaining tumor cell survival.18 USP28 was shown to (unpublished data). This observation further supports UPS37 in stabilize oncoprotein, which is highly expressed in human the PLZF regulation. In addition, USP37 expression was found to colon and breast carcinomas.19 In this study, we have identified be elevated in germ cells according to the microarray database USP37 as a DUB for PLZF/RARA by RNAi screening. We demonstrate (http://www.ebi.ac.uk/arrayexpress/). This also raises a possibility that USP37 physically interacts and modulates PLZF/RARA protein that USP37 may be involved in stabilizing PLZF protein in germ stability and further show an important role of USP37 in PLZF/ cells for spermatogonial stem cell maintenance. RARA-mediated transformation of hematopoietic progenitor cells. In addition to USP37, at least, four other DUBs, including USP2, Thus, our findings not only uncover a new substrate and function of USP29, OTUD6A, and OTUD7B, are capable of regulating PLZF/ USP37 but also provide a strategy in antagonizing PLZF/RARA- RARA level in cells (Figure 1c). The regulation of PLZF/RARA level elicited APL. Small molecules acting as ubiquitin protease inhibitors by these four DUBs could directly or indirectly target PLZF/RARA against specific DUBs have successfully been identified and proved protein for ubiquitination. Although the distribution of USP2 and to effectively alter cellular functions.20,21 Thus, development of OTUD7B is not mainly localized in the nuclear compartment, we small molecules inhibiting USP37 deubiquitinating activity may cannot exclude the possibility that both factors also play provide more effective and reliable clinical therapy for PLZF/RARA- important roles for modulating PLZF/RARA protein level. Similarly, associated APL. we showed that USP29 modulates PLZF/RARA protein level via A recent study has reported that USP37 promotes the G1-S RARA portion. This finding also creates an opportunity in fine- transition via regulating cyclin A stability.22 USP37 deubiquitinates tuning PLZF/RARA level in APL cells, in additional to USP37. cyclin A, causing an increased level of cyclin A to further augment In summary, we demonstrate that USP37 is required for the cyclin A-CDK2 complex formation. Cyclin A-CDK2 then protein stabilization and cell transformation of PLZF/RARA, thus

Oncogene (2012), 1 – 9 & 2012 Macmillan Publishers Limited USP37 deubiquitinates PLZF/RARA W-C Yang and H-M Shih 7

Figure 5. Knockdown of Usp37 attenuates PLZF/RARA-mediated gene suppression and cell transformation. (a) Real-time qPCR analyses of endogenous Cebpa, Cebpb and Cebpe expression in mouse hematopoietic progenitor cells expressing PLZF/RARA or empty vector MSCV. Gel image and western blots show PLZF/RARA expression in retrovirus-infected mouse hematopoietic progenitor cells. Data represent the relative expression of indicated genes. Error bars are mean±s.d. from three experiments performed in duplicate. (b) Real-time qPCR analyses of endogenous Usp37, Cebpa, Cebpb and Cebpe in PLZF/RARA-transduced mouse hematopoietic progenitor cells expressing indicated shRNAs. Error bars are mean±s.d. from three experiments performed in duplicate. (c) Bar graph represents the colony number formed from methylcellulose medium culture of mouse hematopoietic progenitor cells transduced with MSCV vector or MSCV-PLZF/RARA in combination with shLuc or shUsp37#2. Error bars indicate s.d. from three independent experiments. CFU: colony-formation unit. (d) Representative images of colonies formation from transduced mouse hematopoietic progenitor cells in methylcellulose medium at second-round replating. Bar, 200 mm. (e) Giemsa staining of indicated transduced mouse hematopoietic progenitor cells from second-round replating of methylcellulose culture. Bar, 20 mm. (f) FACS analysis of surface marker expression of indicated transduced mouse hematopoietic progenitor cells from second- round replating. Data are representative of three independent experiments. providing USP37 as a potential target for the development of actin (Millipore, Billerica, MA, USA), anti-GFP (Santa Cruz Biotechnology, specific inhibitor in treatment of PLZF/RARA-associated APL. Santa Cruz, CA, USA), anti-Myc (LTK BioLaboratories, Taoyuan, Taiwan), anti-Ub (a gift from Dr. Sheng-Chung Lee), and Anti-Flag and anti-HA beads (Sigma-Aldrich) for Western blotting and immunoprecipitation as MATERIALS AND METHODS where indicated. All of the shRNA constructs against DUBs and luciferase Antibodies and plasmid constructs were obtained from the RNAi consortium at Academia Sinica. The pLKO- The following primary antibodies were used: anti-USP37 (Bethyl Labora- AS3-TetOn-Neo construct (the RNAi consortium, Academia Sinica) was tories, Montgomery, TX, USA), anti-PLZF (Merck, Whitehouse Station, NJ, used to generate stable TetOn-U937 cells by lentiviral infection. For the USA), anti-Flag (Sigma-Aldrich, St Louis, MO, USA), anti-HA (Covance, functional RNAi screening, the complementary DNA (cDNA) coding EGFP- Princeton, NJ, USA), anti-tubulin (Epitomics, Burlingame, CA, USA), anti- PLZF/RARA was cloned into pLKO-AS3W-Tet-TRE-tRFP vector (the RNAi

& 2012 Macmillan Publishers Limited Oncogene (2012), 1 – 9 USP37 deubiquitinates PLZF/RARA W-C Yang and H-M Shih 8 consortium, Academia Sinica) and was expressed in TetOn-U937 cells by retrovirus were concentrated by ultra-centrifugation with 20 000 rpm for lentiviral infection. PLZF/RARA cDNA was separately constructed into 2 h at 4 oC. Mouse hematopoietic progenitor cells co-cultured with OP9 pLKO-AS4.1W-Tet-Hyg (the RNAi consortium, Academia Sinica) and feeder were infected by retrovirus carrying PLZF/RARA construct with pMSCVneo for lentivirus-expressing Flag-tagged PLZF/RARA in TetOn- addition of 0.8 mg/ml polybrene (Sigma-Aldrich). After 16 h infection, U937 and for retrovirus-expressing PLZF/RARA in mouse hematopoietic culture medium was replaced by fresh differentiation culture medium, progenitor cells, respectively. The pLKO.1-mouse Usp37 shRNA lentiviral which is IMDM supplemented with 10% FBS, 20 ng/ml SCF, 10 ng/ml of constructs expressing target sequences are (#1): 50-CGCCTAATGTTGACTT each IL3, IL6 and GM-CSF. For serial infection, the resulting cells were TACAA-30, and (#2): 50-GCAGAAGATGATATTCCAGAA-30. The cDNA con- further infected with lentivirus-expressing indicated shRNA for additional structs of USP29, OTUD5, MPND and OTUD6A were purchased from Open 16 h and replaced with fresh differentiation culture medium. After Biosystems, while USP37, OTUD6B, OTUD7B and USP2 cDNAs were kindly selection by G418 (500 mg/ml) and puromycin (1 mg/ml), cells were gifted from Dr J Wade Harper.13 The cDNAs of USP2, USP29, USP37, OTUD5, subjected for quantitative real time PCR analyses. For replating assay, OTUD6B, OTUD7B, MPND and PLZF/RARA were cloned into pCMV-3xFLAG transduced mouse hematopoietic progenitor cells (10 000 cells/ml) were vector for Flag-tagged protein expression. The cDNAs of OTUD6A and PLZF/ then plated to MethoCult (STEMCELL Technology, Vancouver, BC, Canada) RARA were inserted into pcDNA3-HA vector for HA-tagged protein expression. medium supplemented with 20 ng/ml SCF, 10 ng/ml of each IL3, IL6, and Ubiquitin cDNA was cloned into pCMV-3xMyc vector. USP37 and PLZF/RARA GM-SCF, and 1 mg/ml G418 and 2 mg/ml puromycin for selection. After 7 cDNAs were cloned into pMAL-c2X and pGEX-4T-2 for MBP and GST fusion days, cells were replated for additional 7 days and then the number of protein production, respectively. The catalytically inactive USP37 (USP37 colony in each condition was counted under light microscope. Cellular C350A) mutant was generated by site-directed mutagenesis, as described morphology was analyzed by Giemsa staining of cytospin from the previously.31 The USP37 fragment cDNAs encoding 1–700, 301–979 and 1–300 second-round replating cells. The expression levels of cell surface marker amino-acid residues were cloned into pCMV-3xFLAG vector. were analyzed by FACS with fluorochrome-conjugated antibodies (c-kit/ CD117, clone 2B8; Mac-1/CD11b, clone M1/70; Gr-1/Ly-6G, clone RB6-8C5; all purchased from BioLegend, San Diego, CA, USA). Cell culture, transfection, immunoprecipitation and western analyses HEK-293T and GP2-293 cells were maintained in DMEM with 10% FBS Quantification of PLZF/RARA-regulated gene level (Gibco, Life Technologies, Grand Island, NY, USA). U937, HL60 and NB4 cells Total cellular RNAs were extracted by TRIzol reagent (Invitrogen, Life were maintained in RPMI1640 with 10% FBS (Gibco). OP9 cells were Technologies, Grand Island, NY, USA), and RNA of each sample was maintained in a-MEM with 20% FBS (Hyclone, Thermo Scientific, Austin, TX, subsequently reverse transcribed using ThermoScript reverse transcription- USA) and 60 mM 2-mercaptoethanol, and were served as feeder layer after PCR system (Invitrogen, Life Technologies), according to the manufac- 4-hour treatment with 10 mg/ml mitomycin C (Sigma-Aldrich). Mouse bone turer’s instruction. Reverse transcription PCR product was used for marrow cells were obtained from femurs of combined two male C57BL/6 quantitative real-time PCR analyses (Applied Biosystems 7500 Life mice at 8 weeks of age. Mouse hematopoietic progenitor cells were Technologies, Grand Island, NY, USA) with specific primers, as following: purified from bone marrow cells by negative selection with magnetic Cebpa forward 50-AGGAACTTGAAGCACAAT-30 and reverse 50-ACACAGA beads, according to the manufacturer’s instruction (R&D Systems, GACCAGATACA-30; Cebpb forward 50-CGGGGTTGTTGATGTTTT-30 and Minneapolis, MN, USA) and short-term expanded by co-culture with OP9 reverse 50-CATACGCCTCTTTTCTCATAG-30; Cebpe forward 50-CAAGAAGG feeder layer in IMDM containing 10% FBS (Hyclone), 60 mM 2-mercap- CAGTGAACAA-30 and reverse 50- GCTGAGTCTCCATAATGC-30; Usp37 toethanol, 20 ng/ml murine Scf, 20 ng/ml murine Tpo and 20 ng/ml murine forward 50-CTCATCAGTGTTGTCAGT-30 and reverse 50-TCCAGGTCATTG Flt-3 ligand. All cytokines were purchased from Peprotech (Rocky Hill, NJ, TAAGTG-30; Hprt forward 50-GATTAGCGATGATGAACCAGGTT-30 and reverse USA). Calcium phosphate method was performed for transient transfection 50-CCTCCCATCTCCTTCATGACA-30. Hprt gene expression was used as an of HEK-293T cells. Immunoprecipitation and western analyses were internal control for normalization. The RT-PCR product was used for performed, as described previously.32 In brief, transfected HEK-293T cells semiquantitative PCR analyses with specific primers as following: PLZF/ were harvested in NP40 lysis buffer (50 mM Tris–HCl pH 7.5, 5 mM EDTA, 1% RARA forward 50-TGAAGACGTACGGGTGCGAG-30 and reverse 50-TGTA NP40 and 150 mM NaCl) supplemented with 5 mM NEM and protease GATGCGGGGTAGAGGG-30; actin forward 50-CCTAGAAGCATTTGCGGTGG-30 inhibitor cocktail (Sigma-Aldrich). Cell lysates were immunoprecipitated and reverse 50-GAGCTACGAGCTGCCTGACG-30. The PCR products were with agarose beads conjugated with anti-Flag or anti-HA antibody for 2 h then resolved by 1.5% agarose gel containing ethidium bromide. at 4 oC. For co-immunoprecipitation experiments of endogenous USP37 with PLZF/RARA, TetOn-U937 cells conditionally expressing Flag-tagged PLZF/RARA were harvested by NP40 lysis buffer, incubated with anti-USP37 Statistical analysis or anti-Flag antibody for 16 h at 4 oC and followed by adding magnetic Statistical analyses were carried out by using SAS 9.1.2 (SAS institute Inc, protein G Sepharose beads (GE Healthcare, Waukesha, WI, USA) for Cary, NC, USA) with two-tailed student t test. Two-tailed student t test was additional 1 h. Both resulting beads were washed and subjected to western used here to calculate the EGFP/tRFP intensity ratio between experimental analysis with .specific antibody and control groups. To eliminate false-positive rate occurred from multiple testing, we introduced Bonferroni’s adjustment to correct P-values obtained from t-test, therefore, data with P-valueo0.0001 was considered GST pull-down and deubiquitination assays as statistically significant. GST pull-down assay was performed, as described.32 Two mgof recombinant GST or GST-PLZF/RARA was incubated with 2 mg of MBP- USP37 and immobilized beads (Thermo Scientific) in binding CONFLICT OF INTEREST buffer (10 mM HEPES pH 7.5, 0.5 mM DTT, 0.5 mM EDTA, 0.1% NP-40, and The authors declare no conflict of interest. o 50 mM NaCl) for 4 h at 4 C. After washing three times, the samples were then subjected to western analysis. For in vitro deubiquitination assay, poly-ubiquitinated PLZF/RARA proteins immunoprecipitated from HEK- ACKNOWLEDGEMENTS 293T cells expressing Flag-tagged PLZF/RARA and Myc-tagged Ub were We thank Dr. J Wade Harper for USP37, OTUD6B and USP2 cDNA constructs, Dr incubated with recombinant USP37 protein in 100 ml of deubiquitination o Sheng-Chung Lee for anti-ubiquitin antibody, and Dr Yen-Sung Huang for mouse buffer (50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 1 mM DTT) for 2 h at 37 C. preparation. We also thank for the NRPGM Core RNAi by providing lentiviral The deubiquitination reaction was stopped by adding SDS sampling buffer constructs for TetOn system and shRNAs of DUBs. We are grateful to Dr Michael J and bound proteins were extracted from beads for western analysis. Matunis for comments on the paper. The work was supported by NSC Grants (NSC100-2321-B-001-004; NSC100-3112-B-001-004) and an Academia Sinica Investi- Viral infection and replating assay gator Award to H-M Shih. Lentiviral supernatants were prepared, as described previously.31 In brief, 12 mg lentiviral DNA construct, 3 mg pMD2.G (addgene, Cambridge, MA, USA) and 9 mg psPAX2 (addgene) were cotransfected in HEK-293T cells REFERENCES with 60% confluency in 10-cm dish. Likewise, retroviral supernatants were 1 Mistry AR, Pedersen EW, Solomon E, Grimwade D. The molecular pathogenesis of prepared from 60% confluent GP2-293 cells in 10-cm dish cotransfected acute promyelocytic leukaemia: implications for the clinical management of the with 9 mg pVSV-G and 12 mg retroviral DNA constructs. Both lentivirus and disease. Blood Rev 2003; 17: 71–97.

Oncogene (2012), 1 – 9 & 2012 Macmillan Publishers Limited USP37 deubiquitinates PLZF/RARA W-C Yang and H-M Shih 9 2 Melnick A, Licht JD. Deconstructing a disease: RARalpha, its fusion partners, and 19 Popov N, Wanzel M, Madiredjo M, Zhang D, Beijersbergen R, Bernards R et al. The their roles in the pathogenesis of acute promyelocytic leukemia. Blood 1999; 93: ubiquitin-specific protease USP28 is required for MYC stability. Nat Cell Biol 2007; 3167–3215. 9: 765–774. 3 Guibal FC, Alberich-Jorda M, Hirai H, Ebralidze A, Levantini E, Di Ruscio A et al. 20 Colland F, Formstecher E, Jacq X, Reverdy C, Planquette C, Conrath S et al. Identification of a myeloid committed progenitor as the cancer-initiating cell in Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and acti- acute promyelocytic leukemia. Blood 2009; 114: 5415–5425. vates p53 in cells. Mol Cancer Ther 2009; 8: 2286–2295. 4 Nasr R, Guillemin MC, Ferhi O, Soilihi H, Peres L, Berthier C et al. Eradication of 21 Lee BH, Lee MJ, Park S, Oh DC, Elsasser S, Chen PC et al. Enhancement of acute promyelocytic leukemia-initiating cells through PML-RARA degradation. Nat proteasome activity by a small-molecule inhibitor of USP14. Nature 2010; 467: Med 2008; 14: 1333–1342. 179–184. 5 Nasr R, Lallemand-Breitenbach V, Zhu J, Guillemin MC, de The H. Therapy-induced 22 Huang X, Summers MK, Pham V, Lill JR, Liu J, Lee G et al. Deubiquitinase USP37 is PML/RARA proteolysis and acute promyelocytic leukemia cure. Clin Cancer Res activated by CDK2 to antagonize APC(CDH1) and promote S phase entry. Mol Cell 2009; 15: 6321–6326. 2011; 42: 511–523. 6 Wang ZY, Chen Z. Acute promyelocytic leukemia: from highly fatal to highly 23 Muller C, Yang R, Park DJ, Serve H, Berdel WE, Koeffler HP. The aberrant curable. Blood 2008; 111: 2505–2515. fusion proteins PML-RAR alpha and PLZF-RAR alpha contribute to the 7 Lallemand-Breitenbach V, Jeanne M, Benhenda S, Nasr R, Lei M, Peres L et al. overexpression of cyclin A1 in acute promyelocytic leukemia. Blood 2000; 96: Arsenic degrades PML or PML-RARalpha through a SUMO-triggered RNF4/ubi- 3894–3899. quitin-mediated pathway. Nat Cell Biol 2008; 10: 547–555. 24 Yang R, Nakamaki T, Lubbert M, Said J, Sakashita A, Freyaldenhoven BS et al. 8 Zhang XW, Yan XJ, Zhou ZR, Yang FF, Wu ZY, Sun HB et al. Arsenic trioxide Cyclin A1 expression in leukemia and normal hematopoietic cells. Blood 1999; 93: controls the fate of the PML-RARalpha oncoprotein by directly binding PML. 2067–2074. Science 2010; 328: 240–243. 25 Ji P, Agrawal S, Diederichs S, Baumer N, Becker A, Cauvet T et al. Cyclin A1, the 9 Glickman MH, Ciechanover A. The ubiquitin-proteasome proteolytic pathway: alternative A-type cyclin, contributes to G1/S cell cycle progression in somatic destruction for the sake of construction. Physiol Rev 2002; 82: 373–428. cells. Oncogene 2005; 24: 2739–2744. 10 Ventii KH, Wilkinson KD. Protein partners of deubiquitinating enzymes. Biochem J 26 Doulatov S, Notta F, Rice KL, Howell L, Zelent A, Licht JD et al. PLZF is a regulator 2008; 414: 161–175. of homeostatic and cytokine-induced myeloid development. Genes Dev 2009; 23: 11 Deshaies RJ, Joazeiro CA. RING domain E3 ubiquitin ligases. Annu Rev Biochem 2076–2087. 2009; 78: 399–434. 27 Chen Z, Brand NJ, Chen A, Chen SJ, Tong JH, Wang ZY et al. Fusion between a 12 Nijman SM, Luna-Vargas MP, Velds A, Brummelkamp TR, Dirac AM, Sixma TK et al. novel Kruppel-like zinc finger gene and the retinoic acid receptor-alpha locus due A genomic and functional inventory of deubiquitinating enzymes. Cell 2005; 123: to a variant t(11;17) translocation associated with acute promyelocytic leukaemia. 773–786. EMBO J 1993; 12: 1161–1167. 13 Sowa ME, Bennett EJ, Gygi SP, Harper JW. Defining the human deubiquitinating 28 Shaknovich R, Yeyati PL, Ivins S, Melnick A, Lempert C, Waxman S et al. The enzyme interaction landscape. Cell 2009; 138: 389–403. promyelocytic leukemia zinc finger protein affects myeloid cell growth, differ- 14 Lin RJ, Sternsdorf T, Tini M, Evans RM. Transcriptional regulation in acute pro- entiation, and apoptosis. Mol Cell Biol 1998; 18: 5533–5545. myelocytic leukemia. Oncogene 2001; 20: 7204–7215. 29 Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD et al. Plzf is 15 Duprez E, Wagner K, Koch H, Tenen DG. C/EBPbeta: a major PML-RARA-responsive required in adult male germ cells for stem cell self-renewal. Nat Genet 2004; 36: gene in retinoic acid-induced differentiation of APL cells. EMBO J 2003; 22: 647–652. 5806–5816. 30 Costoya JA, Hobbs RM, Barna M, Cattoretti G, Manova K, Sukhwani M et al. 16 Lavau C, Szilvassy SJ, Slany R, Cleary ML. Immortalization and leukemic transfor- Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet mation of a myelomonocytic precursor by retrovirally transduced HRX-ENL. EMBO 2004; 36: 653–659. J 1997; 16: 4226–4237. 31 Chang CC, Naik MT, Huang YS, Jeng JC, Liao PH, Kuo HY et al. Structural and 17 Li M, Brooks CL, Kon N, Gu W. A dynamic role of HAUSP in the p53-Mdm2 functional roles of Daxx SIM phosphorylation in SUMO paralog-selective binding pathway. Mol Cell 2004; 13: 879–886. and apoptosis modulation. Mol Cell 2011; 42: 62–74. 18 Schwickart M, Huang X, Lill JR, Liu J, Ferrando R, French DM et al. Deubiquiti- 32 Lin DY, Huang YS, Jeng JC, Kuo HY, Chang CC, Chao TT et al. Role of nase USP9X stabilizes MCL1 and promotes tumour cell survival. Nature 2010; 463: SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and 103–107. repression of sumoylated transcription factors. Mol Cell 2006; 24: 341–354.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

& 2012 Macmillan Publishers Limited Oncogene (2012), 1 – 9